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Journal of Clinical Microbiology, April 2008, p. 1192-1199, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02235-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid Identification and Differentiation of Trichophyton Species, Based on Sequence Polymorphisms of the Ribosomal Internal Transcribed Spacer Regions, by Rolling-Circle Amplification{triangledown}

Fanrong Kong,1,{dagger} Zhongsheng Tong,1,2,{dagger} Xiaoyou Chen,1,3,{dagger} Tania Sorrell,1 Bin Wang,4 Qixuan Wu,1 David Ellis,5 and Sharon Chen1*

Centre for Infectious Diseases and Microbiology, The University of Sydney, Westmead Hospital, Westmead, New South Wales, Australia,1 Research Laboratory for Infectious Skin Diseases, Department of Dermatology, Wuhan First Hospital, Wuhan, People's Republic of China,2 Department of Tuberculosis, Beijing Tuberculosis and Thoracic Tumour Research Institute, Beijing, People's Republic of China,3 Retroviral Genetics Laboratory, Centre for Virus Research, Westmead Millennium Institute, The University of Sydney, Sydney, New South Wales, Australia,4 Mycology Unit, Women's and Children's Hospital, Adelaide, South Australia, Australia5

Received 19 November 2007/ Returned for modification 31 December 2007/ Accepted 22 January 2008

DNA sequencing analyses have demonstrated relatively limited polymorphisms within the fungal internal transcribed spacer (ITS) regions among Trichophyton spp. We sequenced the ITS region (ITS1, 5.8S, and ITS2) for 42 dermatophytes belonging to seven species (Trichophyton rubrum, T. mentagrophytes, T. soudanense, T. tonsurans, Epidermophyton floccosum, Microsporum canis, and M. gypseum) and developed a novel padlock probe and rolling-circle amplification (RCA)-based method for identification of single nucleotide polymorphisms (SNPs) that could be exploited to differentiate between Trichophyton spp. Sequencing results demonstrated intraspecies genetic variation for T. tonsurans, T. mentagrophytes, and T. soudanense but not T. rubrum. Signature sets of SNPs between T. rubrum and T. soudanense (4-bp difference) and T. violaceum and T. soudanense (3-bp difference) were identified. The RCA assay correctly identified five Trichophyton species. Although the use of two "group-specific" probes targeting both the ITS1 and the ITS2 regions were required to identify T. soudanense, the other species were identified by single ITS1- or ITS2-targeted species-specific probes. There was good agreement between ITS sequencing and the RCA assay. Despite limited genetic variation between Trichophyton spp., the sensitive, specific RCA-based SNP detection assay showed potential as a simple, reproducible method for the rapid (2-h) identification of Trichophyton spp.

* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology, Westmead Hospital, Darcy Road, Westmead, New South Wales 2145, Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: sharon.chen{at}swahs.health.nsw.gov.au

{triangledown} Published ahead of print on 30 January 2008.

{dagger} F.K., Z.T., and X.C. contributed equally to this study.

Journal of Clinical Microbiology, April 2008, p. 1192-1199, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02235-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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